GSM6321078: 90 week old mouse, replicate 1-2, snRNA-seq; Mus musculus; RNA-Seq
Female mice aged 4 weeks old or 90 weeks old were anesthetized with isofluorane and then acutely decapitated. Their brains were quickly harvested and cut into hemispheres and each hemisphere was frozen immediately on dry ice Optimal Cutting Temperature Compound (Fisher HealthCare) and then stored at –80oC until sectioning. Brains were taken from storage at –80oC and warmed to –18oC in a cryostat (Leica) for 20 minutes before sectioning.  Sections were discarded until the beginning of frontal cortex was apparent. Brains were then blocked on the cryostat using a razor blade to a region containing frontal cortex and striatum. 100 µm coronal sections were then collected approximately from A/P +2 mm to A/P +1 mm, relative to bregma. The resulting sections were collected in an Eppendorft tube and stored at –80oC until snRNA-seq library preparation.   For snRNA-seq library preparation, nuclei were dounced in Nuclei EZ Prep nuclei extraction buffer (Sigma) + 1% RNase Inhibitor. Nuclei were then spun down, filtered through a 70 µm filter, stained with DAPI, and sorted on a FACS machine (BD FacsAria) to separate nuclei from debris. The resulting clean nuclei preparation were then counted and encapsulated on a 10X Genomics Chromium machine, using the 3' Transcriptome V3.1 kit (10X Genomics). After encapsulation, the resulting libraries were reverse transcribed, amplified as cDNA, fragmented, and amplified as a final library following the manufacturer's instructions. The resulting libraries were sequenced on a NovaSeq S4 flowcell (Illumina) to a target depth of ~50,000 reads per nucleus. snRNA-seq